The changing landscape of membrane protein structural biology through developments in electron microscopy

Membrane proteins are ubiquitous in biology and are key targets for therapeutic development. Despite this, our structural understanding has lagged behind that of their soluble counterparts. This review provides an overview of this important field, focusing in particular on the recent resurgence of electron microscopy (EM) and the increasing role it has to play in the structural studies of membrane proteins, and illustrating this through several case studies. In addition we examine some of the challenges remaining in structural determination, and what steps are underway to enhance our knowledge of these enigmatic proteins

The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility

Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K⁺ and Cl⁻ channels now being considered the most active during tumorigenesis. Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers. Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily because of the expression of two early regulatory proteins termed small and large tumor antigens (ST and LT, respectively). Several molecular mechanisms have been attributed to MCPyV-mediated cancer formation but, thus far, no studies have investigated any potential link to cellular ion channels. Here we demonstrate that Cl⁻ channel modulation can reduce MCPyV ST-induced cell motility and invasiveness. Proteomic analysis revealed that MCPyV ST up-regulates two Cl⁻ channels, CLIC1 and CLIC4, which when silenced, inhibit MCPyV ST-induced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes. Consistent with these data, we confirmed that CLIC1 and CLIC4 are up-regulated in primary MCPyV-positive MCC patient samples. We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation. Given the intense interest in ion channel modulating drugs for human disease. This highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC

The mechanosensitive channel YbiO has a conductance equivalent to the largest gated-pore

Bacterial mechanosensitive channels are divided into large (MscL) and small (MscS-like) conductance families. The function of MscS and MscL is to protect cells against osmotic shock by acting as pressure safety valves. Within the MscS-like family, E. coli encodes much larger channels, such as YbiO, MscK, and MscM, but their physiological role remains unclear. Compared to MscL their conductances are reported as 3–10 times lower. We show that YbiO can achieve a conductance of ∼3 nS, and an equivalent pore opening of > 25 Å in diameter, equaling the known largest gated pore, MscL. We determine a cryoelectron microscopy (cryo-EM) structure of YbiO in a sub-open conformation, demonstrating the existence of multiple substates. One substate is consistent with the pore opening extent of our structure and the other matches states previously thought to resemble full openings. Our findings demonstrate surprising capabilities, hinting at new physiological roles for YbiO and potentially other MscS-like channels

Kv1.3-induced hyperpolarization is required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication

Funding: This work was supported by a Rosetrees trust PhD studentship, M662, White Rose BBSRC Doctoral training Partnership in Mechanistic Biology (95519935), BBSRC project grant (BB/T00021X/1), and a Royal Society University Research Fellowship [g:480764 (to J.M.)].Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.Peer reviewe

Author response: Affimer proteins are versatile and renewable affinity reagents

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications

Direct endosomal acidification by the outwardly rectifying CLC-5 Cl-/H+ exchanger

The voltage-gated Cl- channel (CLC) family comprises cell surface Cl- channels and intracellular Cl-/H+ exchangers. CLCs in organelle membranes are thought to assist acidification by providing a passive chloride conductance that electrically counterbalances H+ accumulation. Following recent descriptions of Cl-/H+ exchange activity in endosomal CLCs we have re-evaluated their role. We expressed human CLC-5 in HEK293 cells, recorded currents under a range of Cl- and H+ gradients by whole-cell patch clamp, and examined the contribution of CLC-5 to endosomal acidification using a targeted pH-sensitive fluorescent protein. We found that CLC-5 only conducted outward currents, corresponding to Cl- flux into the cytoplasm and H+ from the cytoplasm. Inward currents were never observed, despite the range of intracellular and extracellular Cl- concentrations and pH used. Endosomal acidification in HEK293 cells was prevented by 25 μm bafilomycin-A1, an inhibitor of vacuolar-type H+-ATPase (v-ATPase), which actively pumps H+ into the endosomal lumen. Overexpression of CLC-5 in HEK293 cells conferred an additional bafilomycin-insensitive component to endosomal acidification. This effect was abolished by making mutations in CLC-5 that remove H+ transport, which result in either no current (E268A) or bidirectional Cl- flux (E211A). Endosomal acidification in a proximal tubule cell line was partially sensitive to inhibition of v-ATPase by bafilomycin-A1. Furthermore, in the presence of bafilomycin-A1, acidification was significantly reduced and nearly fully ablated by partial and near-complete knockdown of endogenous CLC-5 by siRNA. These data suggest that CLC-5 is directly involved in endosomal acidification by exchanging endosomal Cl- for H+. © 2010 The Authors. Journal compilation © 2010 The Physiological Society

Epilepsy-causing <i>KCNT1</i> variants increase K_Na1.1 channel activity by disrupting the activation gate

AbstractGain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.</jats:p